Phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (Ptdlns(4,5)P2 or PIP2) regulate many if not all Transient Receptor Potential (TRP) channels. We will study the regulation of the cold-sensitive TRPM8 and the warmth-sensitive TRPV1 by phosphoinositides. We will focus on elucidating the mechanism and physiological importance of the regulation of these two channels by these lipids. TRPM8 and TRPV1 are expressed in sensory neurons and play important roles in thermosensation, as well as nociception. In Aim 1 we will study the regulation of TRPV1 by phosphoinositides. Our preliminary data and results from others indicate that PIP2 exerts dual control over TRPV1 channels, showing either inhibitory or stimulatory effects in certain settings. Our main hypothesis is that depletion of PIP2 (and PIP) by the activation of various PLC isoforms have differential effects on the channel, depending on the extent of depletion. To test this hypothesis we will combine novel and well established techniques in expression systems, as well as study TRPV1 in sensory neurons. In Aim 2 we will examine the physiological role of PIP2 in the regulation of TRPM8 currents. We have shown that PIP2 activates TRPM8 and that selective depletion of PIP2 is sufficient to inhibit these channels. Our hypothesis is that Ca2+ mediated activation of PLC and the ensuing depletion of PIP2 leads to desensitization of TRPM8 currents. TRPM8 is also inhibited by activators of protein kinase C (PKC), thus PKC mediated inhibition of TRPM8 may also underlie its desensitization. To elucidate the interplay between these two alternative mechanisms, and to define their relative contributions to desensitization, we will use the following approaches. We will test inhibitors and activators of PKC in conjunction with increasing or decreasing PIP2 using various tools, and measure TRPM8 desensitization. Experiments will be performed both in a mammalian expression system and native DRG neurons. In Aim 3 we will study the regulation of TRPM8 in a lipid bilayer system. The plasma membrane is a complex system consisting of a mixture of phospholipids and proteins, where we have limited tools to control the lipid composition of the membrane. Also, in a cellular membrane direct effects of regulatory molecules are hard to differentiate from indirect effects through other proteins. To overcome these limitations, we have purified the TRPM8 protein, incorporated it into planar lipid bilayers, and showed that it exhibits menthol-activated PIP2- dependent activity. We also reconstituted TRPM8 from mammalian cells expressing TRPM8, via native membrane vesicles. These systems allow full control of the phospholipid content of the membrane, therefore serves as a unique tool to study fundamental questions in lipid gating of TRPM8 channels that could not be answered with other techniques. Our data will provide mechanistic insight into the regulation of temperature sensitive TRP channel, and may also serve as a basis for better local pain control.